Method for identifying compound for inhibiting an activity of a histone lysine demethylase

ABSTRACT

The present invention relates to a method for identifying a compound that inhibits an activity of a histone lysine demethylase.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a non-provisional utility application which claims priority to U.S. Provisional Application No. 61/955,225, filed on Mar. 19, 2014, incorporated herein by reference in its entirety. The sequence listing text file, file name 2118_WWJ_SEQ, created Mar. 18, 2015, file size 38,571 bytes, is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a method for identifying a compound that inhibits an activity of a histone lysine demethylase.

DESCRIPTION OF PRIOR ART

Histone lysine demethylases (KDMs) that regulate a dynamic, reversible status of “methyl” histone codes have gained much attention. Mutations, amplifications, deletions and aberrant expression of KDMs have been identified in a variety of cancers and their roles in modulating the behaviours of cancer cells have been substantiated. As such, increasing attention has been paid to evaluate KDMs as potential therapeutic targets for cancer. There are now 8 KDM families including 28 members identified. KDM2-KDM8 constitute a large superfamily that shares a Jumonji C (JmjC) domain which functions as α-ketoglutarate (AKG) and Fe(II)-dependent demethylase. Notably, each family exhibits its exquisite substrate specificity toward different histone lysine residues, thereby effectively integrating the upstream signals and modulating the chromatin conformation.

The largest KDM4 gene family (four paralogs KDM4A-KDM4D and two pseudogenes KDM4E and KDM4F) has been shown to be an “eraser” of a repressive mark H3K9me3/me2, while its subfamily KDM4A-KDM4C also demethylates H3K36me3/me2. KDM4A and KDM4B are over expressed in a variety of cancers including prostate, breast, colorectal, lung, gastric, esophageal, lymphoma, renal cancer, and medulloblastoma. For prostate and breast cancers, this family of demethlases have the added significance in being coactivators of androgen receptor (AR) (KDM4A, B, C and D) and estrogen receptor (ER) (KDM4A and B). They function to stimulate the transcriptional potential of the receptors. KDM4B also regulates the turnover of AR. Given the important roles of AR and ER in prostate and breast carcinogenesis, KDM4A/4B are considered as promising drug targets of intervention in these malignancies.

Thus far, inhibitors for KDM4 proteins described are largely AKG analogues: N-oxalylglycines (OGAs) that inhibit KDM4A, KDM4C and KDM4D, pyridine 2,4-dicarboxylic acids (PD2s) developed based on KDM4E, and 8-hydroxyquinolines (8HQs) of which 5-carboxy-8HQ displays the highest potency on KDM4E in vitro (IC50=0.2 μM). Yet, as a prodrug, cytotoxic IC50 of PD2 is at ˜mM range in cultured cells, due to its poor cell-penetrating ability, while 5-carbxoxy-8HQ exhibits a relatively high cytotoxic IC50 in HeLa cells (86.5 μM).

SUMMARY OF THE INVENTION

The present invention provides a method for identifying a compound which inhibits an activity of a histone lysine demethylase, comprising: (a) using a computer program to generate a three-dimensional structure of a pocket of a histone lysine demethylase, wherein the pocket comprises three sites: alpha-ketoglutarate (AKG), a methylated lysine, and a NIQ; (b) screening for a compound that interacts with the three sites of said pocket; and (c) testing the compound screened in (b) by in vitro or in vivo assay for its ability to inhibit the activity of the histone lysine demethylase, thereby identifying a compound that inhibits the activity of the histone lysine demethylase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows (A) the electron density map for the Ni (II), PD2, and the H3K9me3 peptide. The 2Fo-Fc electron density maps are contoured at 1.0 σ. (B) The JmjC domain of KDM4B folds into a β-barrel structure.

FIG. 2 shows the differential ligand binding region between KDM4A/KDM4B/KDM4C and KDM4D. Superposition of KDM4A, KDM4B, KDM4C, and KDM4D reveals two heterogeneous regions, RKDM and NIQ. Surface representation of KDM4A, KDM4B, and KDM4D shows that the RKDM, NIQ regions and a crucial isoleucine (KDM4A, 171; KDM4B, 172) make several contacts with the peptide at (−2) and (+3, +4) sites in KDM4A and KDM4B, respectively. The corresponding GEAR and HKK sites deviate away from H3K9me3. PDB used in this Figure are: KDM4A (PDB: 20Q6); KDM4B (PDB: 4LXL); KDM4D (PDB: 4HON).

FIG. 3(A) shows demethylation of calf thymus H3 by bacteria-expressed KDM4A (upper panel) and KDM4B (lower panel) determined in the presence of 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene by western blotting analysis. H3 lysine modifications are probed with H3K9me3 and H3K36me3 antisera, respectively. FIG. 3(B) shows inhibition kinetics of KDM4A/4B demethylation activity by 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene. The inset in each panel shows the double reciprocal form, where the 1/relative activity is plotted versus 1/[H3K9me3] at various fixed concentrations of the inhibitor.

FIG. 4(A) shows surface representation of superimposed KDM4B.PD2.H3K9me3 and KDM4B.1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene complexes. PD2 and H3K9me3 are drawn as stick models. 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene is show as ball-and-stick models. AKG site is labeled as site II, methylated lysine site as site I, RKDM as site III and NIQ as site IV. FIG. 4(B) shows schematic representation of interactions between 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene and KDM4B. FIG. 4(C) shows schematic representation of interactions between PD2 and KDM4B. The hydrogen-bonding contacts are shown as broken lines.

FIG. 5 shows (A) interaction profiles between residues from KDM4B and compounds. These compounds are divided into three groups based on their docked locations. The profile contains two interaction types [hydrogen-bonding (H) and van der Waals (V)]. A cell is colored in gray if a compound yields hydrogen-bonding or van der Waals interaction with a residue; otherwise, the cell is colored in black. FIG. 5 shows the docked conformations of (B) NSC107408 (in group 1), (C) NSC15975 (in group 2), and (D) NSC640999 (in group 3). NSC107408, NSC15975, and NSC640999 lack the interactions in the AKG site, the methylated lysine site, and the NIQ site, respectively, compared to 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene.

FIG. 6(A) shows the expression values of KDM4A in normal prostate gland and tumor tissues from the selected studies, which were obtained from Oncomine™ (Compendia Bioscience, Ann Arbor, Mich., USA) database. The box whisker plots show the box encompasses 25th-75th percentile, median as line within the box, and 10th and 90th percentiles as error bars. FIG. 6(B) shows the expression values of KDM4B in normal prostate gland and tumor tissues from the selected studies, which are obtained from Oncomine™ database. The box whisker plots show the box encompasses 25th-75th percentile, median as line within the box, and 10th and 90th percentiles as error bars. P values are determined by one-tailed Student's T-test and calculated based on the comparison of normal vs. cancer; normal vs. primary sites; or normal vs. metastasis. FIG. 6(C) shows the qRT-PCR analysis of KDM4A and KDM4B expression in normal prostate primary cell (PrEC), normal prostate epithelial cell lines (RWPE-1 and PNT2), and prostate cancer cell lines (LNCaP, C4-2, C4-2B, CWR22rv1, CWR-R1, VCaP, DU145 and PC3). Asterisks indicate significant over-expression as compared to normal cells.

FIG. 7(A) shows KDM4A and KDM4B are crucial for growth of LNCaP cells. LNCaP cells are infected with lentivirus with control shRNA (control) and shKDM4A or shKDM4B as indicated (left panel). The qRT-PCR analysis is performed to evaluate the expression of KDM4s (right panel). FIG. 7(B) shows cytotoxicity IC50 (CC50) and inhibition of H3K9me3 by 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene in LNCaP cells. LNCaP cells are treated with 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene for three days, and the viability is measured by viable cell count. SD is derived from biological triplicates. The level of H3K9me3 of 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene-treated cells (24 hr) is detected and quantified by AlphaView SA (Cell Biosciences Inc.). The level of H3K9me3 is shown by bar graph. FIG. 7(C) shows flow cytometric analysis of DNA contents in LNCaP cells treated with DMSO (mock) or 50 μM 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene for 3 days.

FIG. 8(A) shows DAVID functional annotation of the genes that shows two-folds alterations in expression. GO terms associated with the altered genes that show statistically strong enrichment with low P-values are listed. Bar graph and the numbers labelled indicate gene count of each pathway. % of hits indicates the percentage of genes that are altered in each GO. FIG. 8(B) shows expression of tumour suppressors and oncogenes that are up- and down-regulated in the inhibitor treated cell, respectively. FIG. 8(C) shows overlap of the inhibitor-altered genes with androgen responsive genes.

FIG. 9 shows AR-signature genes are differentially expressed in 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene-treated LNCaP cell.

FIG. 10 shows the binding pocket in KDM4s. Strictly conserved residues that bind to Fe(II) (H189, E191 and H276 in KDM4B) and AKG(Y133, N199, K207) are shown (upper left). Superposition of H3K9me3 liganded structures reveals four strictly conserved residues of KDM4s (D136, E170, Y176, and K242 in KDM4B) make contacts with the peptide at R8 (−1), K9, S10 (+1) and T11 (+2) positions. PDB used in the left-top figure are: KDM4A (PDB: 2OQ6); KDM4B (PDB: 4LXL); and KDM4D (PDB: 4HON). H bonds (<3.5 Å) are shown as dash lines.

FIG. 11 shows the analysis of KDM4A and KDM4B that demethylate H3K9me3/me2 and H3K36/me3/me2 of histones but not H3K27me3/me1. Demethylation of calf thymus histones by KDM4A and KDM4B is detected by various antisera as indicated. Histones are incubated without or with KDM4A/KDM4B for a certain time as indicated.

FIG. 12 shows top five results of the self-docking tests for the KDM4B.PD2 complex model. Self-docking test of KDM4B (PDB code: 4LXL) is a complex structure within PD2 and H3K9me3. The top five docking poses are shown as stick. The reference pose is shown as ball-and-stick. Ni is shown as a sphere.

FIG. 13(A) shows that the rest of datasets exhibit higher median values of KDM4A expression in PCa sites, despite no statistical significance. The expression values of KDM4A and KDM4B in normal prostate gland and tumor tissues from the selected studies are obtained from Oncomine™ (Compendia Bioscience, Ann Arbor, Mich., USA) database. The box whisker plots show the box encompasses 25th-75th percentile, median as line within the box, and 10th and 90th percentiles as error bars. FIG. 13(B) shows that the rest of datasets exhibit higher median values of KDM4B expression in PCa sites, despite no statistical significance. The expression values of KDM4A and KDM4B in normal prostate gland and tumor tissues from the selected studies are obtained from Oncomine™ database. The box whisker plots show the box encompasses 25th-75th percentile, median as line within the box, and 10th and 90th percentiles as error bars. P values are determined by one-tailed Student's T-test and calculated based on the comparison of normal vs. cancer; normal vs. primary sites; or normal vs. metastasis.

FIG. 14 is an illustration of linked T01 functional group. When the original CL atom of the R2 group is replaced by the T01 fragment, the Naphthalene-like group generates two cation-pi interaction against the LYS242 residues of KDM4B to stabilize interactions of the receptor-ligand complex.

DETAILED DESCRIPTION OF THE INVENTION

As used herein in the specification, “a” or “an” may mean one or more. As used herein in the claim(s), when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.

The present invention identifies a selective inhibitor 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene (NSC636819) toward KDM4A/4B/4C subfamily. Kinetic and docking analyses reveal crucial binding sites of 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene unique in the KDM4A/KDM4B/KDM4C subfamily. Further, the pharmacological and genetic inhibition of KDM4A/4B significantly lowers the viability of prostate cancer cells, principally due to its potency to inhibit AR transcriptional network.

A method for identifying a compound that modulates the activity of a histone lysine demethylase having a pocket formed by residues including Gln85, Tyr133, Asp136, Tyr176, Tyr178, Phe186, His189, Glu191, Asn199, Lys207, His241, Lys242, His277 and/or Asn291, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 11 or a fragment or derivative thereof; a pocket formed by Gln84, Tyr132, Asp135, Tyr175, Tyr177, Phe185, His188, Glu190, Asn198, Lys206, His240, Lys241, His276 and/or Asn290, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 12 or a fragment or derivative thereof; a pocket formed by Gln86, Tyr134, Asp137, Tyr177, Tyr179, Phe187, His190, Glu192, Asn200, Lys208, His242, Lys243, His278 and/or Asn292, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 13 or a fragment or derivative thereof; a pocket formed by Gln88, Tyr136, Asp139, Tyr179, Tyr181, Phe189, His192, Glu194, Asn202, Lys210, His244, Lys245, His280 and/or Asn294, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 14 or a fragment or derivative thereof; or a pocket formed by Gln85, Tyr133, Asp136, Tyr176, Tyr178, Phe186, His189, Glu191, Asn199, Lys207, His241, Lys242, His277 and/or Asn291, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 15 or a fragment or derivative thereof; the method comprises modeling the compound in the pocket of the histone lysine demethylase; and determining the effect of the compound on the rate or degree of methylation of a substrate of the histone lysine demethylase.

The present invention provides a method for identifying a compound which inhibits an activity of a histone lysine demethylase, comprising: (a) using a computer program to generate a three-dimensional structure of a pocket of a histone lysine demethylase, wherein the pocket comprises three sites: an alpha-ketoglutarate (AKG), a methylated lysine, and a NIQ; (b) screening for a compound that interacts with the three sites of said pocket; and (c) testing the compound screened in (b) by in vitro or in vivo assay for its ability to inhibit the activity of the histone lysine demethylase, thereby identifying a compound that inhibits the activity of the histone lysine demethylase.

As used herein, the histone lysine demethylase (KDM) comprises a KDM4A, a KDM4B, a KDM4C, a KDM4D, a KDM4E. The peptide sequence of the KDM4A is SEQ ID NO: 11. The peptide sequence of the KDM4B is SEQ ID NO: 12. The peptide sequence of the KDM4C is SEQ ID NO: 13. The peptide sequence of the KDM4D is SEQ ID NO: 14. The peptide sequence of the KDM4C is SEQ ID NO: 13. The peptide sequence of the KDM4E is SEQ ID NO: 15.

In one embodiment, the pocket of the histone lysine demethylase is formed by residues including Gln85, Tyr133, Asp136, Tyr176, Tyr178, Phe186, His189, Glu191, Asn199, Lys207, His241, Lys242, His277 and Asn291, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 11 or a fragment or derivative thereof. The alpha-ketoglutarate site is defined as the cavity occupied by alpha-ketoglutarate in the active-site histone lysine demethylase shown in SEQ ID NO: 11. The alpha-ketoglutarate site is surrounded by Y133, F186, H189, E191, S197, N199, K207, W209, T271, H277, and S289 of the SEQ ID NO: 11. The methylated lysine site is defined as the cavity occupied by the methylated lysine in the active site of the histone lysine demethylase shown in SEQ ID NO: 11. The methylated lysine site is enclosed by E170, G171, V172, Y176, Y178, E191, S197, S289, T290 and N291 of the SEQ ID NO: 11. The NIQ site is defined to comprise amino acid residues N87, 172, and Q89 of the SEQ ID NO: 11.

In another embodiment, the pocket of the histone lysine demethylase is formed by residues including Gln84, Tyr132, Asp135, Tyr175, Tyr177, Phe185, His188, Glu190, Asn198, Lys206, His240, Lys241, His276 and Asn290, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 12 or a fragment or derivative thereof. The alpha-ketoglutarate site is defined as the cavity occupied alpha-ketoglutarate in the active site of histone lysine demethylase shown in SEQ ID NO: 12. The alpha-ketoglutarate site is surrounded by Y132, F185, H188, E190, S196, N198, K206, W208, T270, H276, and S288 of the SEQ ID NO: 12. The methylated lysine site is defined as the cavity occupied by the methylated lysine in the active site of the histone lysine demethylase shown in SEQ ID NO: 12. The methylated lysine site is enclosed by E169, G170, V171, Y175, Y177, E190, S196, S288, T289 and N290 of the SEQ ID NO: 12.The NIQ site is defined to comprise amino acid residues N86, 171, and Q88 of the SEQ ID NO: 12.

In one embodiment, the pocket of the histone lysine demethylase is formed by residues including Gln86, Tyr134, Asp137, Tyr177, Tyr179, Phe187, His190, Glu192, Asn200, Lys208, His242, Lys243, His278 and Asn292, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 13 or a fragment or derivative thereof. The alpha-ketoglutarate site is defined as the cavity occupied alpha-ketoglutarate in the active site of histone lysine demethylase shown in SEQ ID NO: 13. The alpha-ketoglutarate site is surrounded by Y134, F187, H190, E192, S198, N200, K208, W210, T272, H278, and S290 of the SEQ ID NO: 13. The methylated lysine site is defined as the cavity occupied by the methylated lysine in the active site of the histone lysine demethylase shown in SEQ ID NO: 13. The methylated lysine site is enclosed by E171, G172, V173, Y177, Y179, E192, S198, S290, T291 and N292 of the SEQ ID NO: 13. The NIQ site is defined to comprise amino acid residues N88, 173, and Q90 of the SEQ ID NO: 13.

In another embodiment, the pocket of the histone lysine demethylase is formed by residues including Gln88, Tyr136, Asp139, Tyr179, Tyr181, Phe189, His192, Glu194, Asn202, Lys210, His244, Lys245, His280 and Asn294, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 14 or a fragment or derivative thereof.

In one embodiment, the pocket of the histone lysine demethylase is formed by residues including Gln85, Tyr133, Asp136, Tyr176, Tyr178, Phe186, His189, Glu191, Asn199, Lys207, His241, Lys242, His277 and Asn291, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO: 15 or a fragment or derivative thereof.

In another embodiment, the activity is the demethylating activity.

The histone lysine demethylase (KDM) has the function for demethylating the histone code. In one embodiment, the histone code comprises a H3K9me3, a H3K9me2, a H3K36me3, and a H3K36me2. In a preferred embodiment, the KDM4A and KDM4B demethylate H3K9me3/me2.

The compounds of the present invention can also be designed by visually inspecting the three-dimensional structure of the KDM to determine more effective inhibitors. This type of modeling is generally referred to as “manual” drug design. Manual drug design can employ visual inspection and analysis using a graphics visualization program. Initially compounds are selected by manual drug design. The structural analog thus designed can then be modified by computer modeling programs to better define the most likely effective candidates. Reduction of the number of potential candidates is useful as it may not be possible to synthesize and screen a countless number of compound variations that may have some similarity to known inhibitory molecules.

In another embodiment, the compound that inhibits the activity of the histone lysine demethylase further is used to treat cancer.

The present invention also provides a method for treating cancer, which comprises administering an effective amount of compound and a pharmaceutically acceptable carrier to a subject in need thereof, wherein the compound has the structure of formula I:

wherein R₁₋₄ is F, Cl, Br, I, At, hydroxyl or C_(x)H_(y)N_(z)O_(α)S_(β) respectively,

wherein x=1-11, y=3-15, z=0-3, α=0-2 and β=0-1.

The present invention may be used to treat, alleviate, ameliorate, relieve, delay onset of, inhibit progression of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition induced by the cancer. In a preferred embodiment, the method of the present invention further treats a prostate cancer.

In one embodiment, the cancer is selected from prostate cancer, breast cancer, colorectal cancer, lung cancer, gastric cancer, esophageal cancer, lymphoma, renal cancer or medulloblastoma. In a preferred embodiment, the cancer is a prostate cancer.

In another embodiment, the compound is a 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene.

A “effective amount” is an amount effective to prevent, lower, stop or reverse the development of, or to partially or totally alleviate the existing symptoms of a particular condition for which the subject being treated.

The “compound” or “1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene” may be formulated for administering via sterile aqueous solution or dispersion, aqueous suspension, oil emulsion, water in oil emulsion, site-specific emulsion, long-residence emulsion, sticky-emulsion, microemulsion, nanoemulsion, liposomes, microparticles, microspheres, nanospheres, nanoparticles, minipumps, and with various natural or synthetic polymers that allow for sustained release. The compounds comprising the NRIP may also be formulated into aerosols, tablets, pills, sterile powders, suppositories, lotions, creams, ointments, pastes, gels, hydrogels, sustained-delivery devices, or other formulations used in drug delivery.

As used herein, the term “pharmaceutically acceptable carriers” are determined in part by the particular composition being administered, as well as by particular method used to administer the composition. As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. The phrase “pharmaceutically-acceptable” refers to molecular entities and compositions that do not produce an allergic or similar untoward reaction when administered to a subject. The preparation of an aqueous composition that contains a protein as an active ingredient is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified.

In one embodiment, the subject is an animal. Preferably, the subject is a mammal. More preferably, the subject is a human.

In one embodiment, the compound inhibits a cancer cell growth by inhibiting an expression of a histone lysine demethylase (KDM). In a preferred embodiment, the compound induces an apoptosis of a cancer cell by inhibiting an expression of the histone lysine demethylase (KDM). In a more preferred embodiment, the compound inhibits the demthylating function of the KDM. In another embodiment, the KDM is a KDM4A or a KDM4B.

In another embodiment, the KDM demethylates a histone. In a preferred embodiment, the histone comprises a H3K9me3, a H3K9me2, a H3K36me3, and a H3K36me2. In a preferred embodiment, the KDM4A and the KDM4B demethylate the H3K9me3 and the H3K9me2.

In one embodiment, the compound further inhibits the cancer cell growth or induces an apoptosis of a cancer cell by inhibiting an androgen receptor (AR), wherein the AR is a coactivator of the KDM4A and the KDM4B.

EXAMPLES

The examples below are non-limiting and are merely representative of various aspects and features of the present invention.

Material and Methods:

Cloning, Expression, and Purification

Human KDM4B 1-348 and KDM4A 1-347 were PCR amplified from chromosomal DNA using forward and reverse primers as follows:

SEQ ID Gene Primer sequence No. KDM4B Forward: 5′-AAA CAT ATG GGG TCT GAG 1 1-348 GAC CAC GGC GCC-3′ (NdeI) Reverse: 5′- AAA AAA CTC GGG GCT CTC 2 GAG CTA CGT GGG CCG -3′ (XhoI) KDM4A Forward: 5′- AAA CAT ATG GCG AGC GAA 3 1-347 AGC GAA ACT CTG -3′ (NdeI) Reverse: 5′- AAA GGA TCC CTA CGT GGG 4 CAG AGT ATG GTC-3′ (BamHI)

PCR was performed with HiFi DNA polymerase kit using a C1000 Touch™ Thermal Cycler (Bio-Rad Laboratories, Inc., USA): initial denaturation, at 95° C. for 5 min followed by 25 cycles of denaturation at 95° C. for 30 s, annealing at 55° C. for 30 s, and extension at 72° C. for 70 s. The amplified product was inserted into pET28a (Novagen, Inc., USA) to generate pET28a-KDM4B 1-348 and pET28a-KDM4A 1-347 which were introduced into Escherichia coli BL21 (DE3) cells. Expression of protein was induced by addition of 0.5 mM isopropyl-β-d-thiogalacto-pyranoside (IPTG) at 16° C. for 21 h. Bacterial pellets were fractionated by sonication to collect soluble proteins in cytosolic fractions. The His6-tagged KDM4B or KDM4A proteins were purified by a nickel affinity column (Ni Sepharose™ High Performance, GE Healthcare) using the elution buffer containing 500 mM NaCl, 250 mM imidazole and 50 mM HEPES (pH 7.5). The protein was concentrated and further purified by a 16/60 Superdex 75 gel filtration column equilibrated with 50 mM HEPES pH 7.5, 500 mM NaCl. The protein purity was analyzed by SDS-PAGE analysis. Protein concentration was assayed by the Bradford method using bovine serum albumin as the standard.

Enzyme Assay

Formaldehyde dehydrogenase-coupled demethylase assay was used to determine the demethylase activity and select the potent inhibitors. All inhibitors were dissolved in dimethyl sulfoxide (DMSO) at various concentrations, and added to the mixture that the final DMSO concentration is 5%. The reagents for demethylase reactions were dissolved in HEPES buffer (50 mM, pH 7.5), with the exception of Fe(II) solutions, which were made using (NH4)2Fe(SO4)2 dissolved in 20 mM HCl to make 400 mM stock solution. All the reagents were stored at −30° C. FDH, NAD+, H-TKQTARK(Me3)STGGKAPR-OH (TR-15, H33-17K9me3, Kelowna), DMSO, and KDM4B were added first to 96-well black immune plate (SPL Life Science) and incubated together on ice for 15 min. Then the plate was put into FLUOStar OPTIMA ELISA reader (BMG LABTECH) with 37° C., and the reaction was started by adding ascorbic acid (ascorbate), Fe(II), and α-ketoglutarate (AKG) to final concentration of 50 mM HEPES, pH 7.5, 2 μM of KDM4B, 5% DMSO, 0.01 U FDH (Sigma), 1 mM NAD+, 1 mM 2-OG, 2 mM ascorbate, and 50 μM Fe(II), various concentration of H3K9me3 peptide, and the final volume was 50 μl. Each reaction was incubated at 37° C. for 30 min and the production of NADH would be detected by using the fluorescence Ex 360/Em 470.

Crystallization

Crystallization was performed by the hanging-drop vapor-diffusion method at 4° C. Equal volumes of a protein sample and the reservoir solution were mixed. Initial crystallization screening was automated using a robot Oryx8 (Douglas Instruments, UK) and the reagents of seven sets of crystallization kits: Crystal Screen I and II kits (Hampton Research), Index kit (Hampton Research), Clear Strategy Screen I and II kits (Molecular Dimension), Wizard kit (Emerald), and JB Screen classic HTS I and II kits (Jena Bioscience). Crystals of KDM4B (10 mg/ml protein, 4 mM PD2 and 5 mM H3K9me3 peptide) were grown in 0.1 M MES (pH 6.5), 0.2 M magnesium acetate, 20% (w/v) polyethylene glycol (PEG) 8000. Optimized crystals used for diffraction (12 mg/ml within 4 mM PD2 and 5 mM H3K9me3 peptide) were grown in 0.1 M MES (pH 6.5), 0.2 M magnesium acetate, 24% (w/v) polyethylene glycol (PEG) 8000. The crystal diffracted to 1.87 Å, belonged to space group P212121, and had unit cell dimensions of a=54.36, b=78.48, c=83.89 Å. The asymmetric unit contained one molecule.

X-ray Data Collection and Processing

Crystals were flash frozen in a stream of liquid nitrogen and then screened and characterized using an RU-300 rotating-anode X-ray generator (Rigaku/MSC Inc., USA) at the Macromolecular X-ray Crystallographic Laboratory of the National Tsing Hua University, Taiwan. The KDM4B.PD2.H3K9me3 dataset was collected at the SPring-8 BL12B2 beamline, Japan, with an ADSC Quantum 4R detector. All datasets were indexed, integrated, and scaled using HKL-2000. Data collection statistics are shown in Table 1.

TABLE 1 Crystallographic Data and Refinement Statistics of KDM4B · PD2 · H3K9me3 Data Collection Beamline SPring-8 BL-12B2^(a) Wavelength (Å) 1.0000 Space group P2₁2₁2₁ Cell dimensions a, b, c (Å) 54.4, 78.5,83.9 α, β, γ (⁰) 90.0, 90.0,90.0 Resolution limit (Å)   30-1.87 Unique reflections 30463 Completeness (%)^(b) 99.4 (98.0) Avg I/σ(I)^(b) 31.2 (6.0)  R_(merge) (%)^(b,c)  4.3 (36.8) Redundancy^(b) 12.5 (11.2) Refinement Resolution limit (Å) 28.67-1.87 Number of atoms 3125 Protein atoms 2768 Solvent atoms 285 Ligand atoms 72 Estimated coordinate error (Å) 0.142 R_(work) ^(d)/R_(free) ^(e) 17.6/22.5 Overall B-factor (Å²) 28.01 RMSD bond lengths (Å)^(f) 0.012 RMSD bond angles (⁰)^(f) 1.39 Ramachandran Analysis (%)^(g) Favored 95.9 Allowed 3.8 Disallowed 0.3 ^(a)BL-12B2 Taiwan beamline at SPring-8, Hyogo, Japan ^(b)Values in parentheses refer to statistics in the highest-resolution shell. ^(c)R_(merge) = Σ|I_(obs) − <I>|/ΣI_(obs). ^(d)R_(work) = Σ|F_(obs) − F_(calc)|/ΣF_(obs), where F_(obs) and F_(calc) are the observed and calculated structure-amplitudes, respectively. ^(e)R_(free) was computed using 5% of the data assigned randomly. ^(f)Root mean square deviation. ^(g)Estimated standard uncertainties based on maximum likelihood.

Structure Determination and Refinement

Crystallographic refinement used the maximum-likelihood target function module in REFMAC5. The KDM4B.PD2.H3K9me3 structures were constructed by MOLREP with the KDM4A (PDB: 2YBS) as the template and were refined using REFMAC5 coupled with ARP/wARP, which automatically added water molecules. The 2Fo-Fc electron density maps were generated by FFT and plotted by PyMOL. The validities of the KDM4B.PD2.H3K9me3 structure were assessed by PROCHECK.

Structural Comparison

The KDM4B structure was compared with protein structures in the DALI server. The structures of KDM4A.H3₁₋₁₇K9me3 (PDB code: 2P5B), KDM4B.PD2 (PDB code: 4LXL; this study), KDM4C.OGA (PDB code: 2XML), KDM4D.AKG.H3₆₋₁₅K9me3 (PDB code: 4HON), and KDM4E (PD2; PDB code: 2W2I) were superimposed by LSQMAN in O. ESPript was used for the combined sequence and secondary structure alignments and Figure preparation. PyMol was used to prepare the figures.

Virtual Screening and Molecular Modeling

The binding site for virtual docking screening of putative inhibitors was prepared by including protein atoms located ≦10-Å-radius sphere centered around the bound ligand of KDM4A (PDB code 2YBK. The present invention utilized GEMDOCK to screen the NCI database (236,962 compounds). Top ranked, available compounds were selected for testing in the KDM4A/KDM4B inhibitory assay.

Molecular Modeling

Discovery Studio v3.0 (Accelrys Inc., USA) was used to prepare, energy minimize, and refine a KDM4B model for molecular dynamics. The default parameters of ChiRotor were used to optimize side-chain conformations. Energies of the protein models were further minimized using CHARMm. 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene was then docked into KDM4B by GEMDOCK, a robust flexible ligand docking tool, was first used in conjunction with its default settings to generate conformations and carry out a docking analysis for ligand-containing KDM4B. Top 10 of compounds ranked by the docking energy were derived. CDOCKER with CHARMm forcefield was used to refine the docked models. To estimate the practicability of the proposed docking procedure, the present invention performed self-docking against the co-crystal KDM4B-PD2 structure (PDB code: 4LXL). Top five docked poses with minimum RMSD≦1 Å were derived (FIG. 12). The average of RMSD for self-docked poses of KDM4B-PD2 is 0.86 Å.

Cell Culture

Primary PrEC cells were purchased from Clonetics (Walkersville, Md.) and cultured in serum-free prostate epithelial cell growth medium following the vendor's directions. Cell lines RWPE1, LNCaP (LNCaP-FGC), CWR22Rv1 (22Rv1), VCaP, DU145, PC3 (all purchased from ATCC), and PNT2 (Sigma Aldrich, Mo.), were cultured under condition as recommended. CWR-R1 cell (40) and LNCaP derived C4-2, C4-2B cells were cultured in RPMI1640 medium containing 10% FBS.

RNA Interference and Quantitative RT-PCR

Lentiviral vector pLKO.1 carrying sequences encoding a shRNA that specifically targets KDM4A and KDM4B gene (TRC library Clone ID TRCN0000234910 and TRCN000018014) were co-transfected with viral packaging plasmids in 293T cells to generate the shRNA lentiviral particles. Empty pLKO.1 plasmid was used as negative control. The lentiviral supernatant was collected after 48-hr transfection and concentrated by Lenti-X Concentrator (Clontech, Calif.). The precipitated viral particle was resuspended in fresh RPMI1640 medium with 10% FBS for subsequent LNCaP infection and transduced into LNCaP cells for 72 hrs. Cells were then harvested and total RNA was isolated, followed by cDNA synthesis and real-time PCR analysis using iQ5 iCycler thermal cycler (Bio-Rad, Calif.). Threshold cycle values were normalized against actin transcript level. Individual samples were performed in triplicate and converted to relative gene expression using QGene96 software. Primer sequences used are as follows:

SEQ ID Gene Primer sequence No. KDM4A Forward: 5′- AGG AGA GTG AAC TGC CTC 5 CA -3′ Reverse: 5′- GGT CTC CTT CCT CTC CAT 6 CC -3′ KDM4B Forward: 5′- TCA CGC AGT ACA ATA TCC 7 AG -3′ Reverse: 5′- TCG TCA TCA TAC AAA GAG 8 CC -3′ actin Forward: 5′- GTA CCA CTG GCA TCG TGA 9 TGG ACT -3′ Reverse: 5′- CCG CTC ATT GCC AAT GGT 10 GAT -3′

Cell Proliferation Assay

LNCaP cell was seeded in 48-well plate one day prior to lentivirus infection. After subjected to the shRNA lentivirus (day 0), cell proliferation was measured every two days by MTT colorimetric assay according to the manufacturer's instruction (Roche, Ind.).

Immunoblotting and Flow Cytometry

Total cell lysates were obtained by lysing the cell with buffer (50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA, protease inhibitors) for 15 min on ice, followed by 10 min of sonication cycle (30 sec on, 30 sec off) on ice. The level of total histone H3 and trimethylated histone H3 Lys9 was analyzed by western blotting using anti-histone H3 and anti-H3K9me3 antibodies (Abcam, Mass.). Cells treated with mock and the inhibitor were harvested and fixed by 70% ethanol for >4 hours at −20° C., followed by propidium iodide (Sigma Aldrich) staining The DNA content was analyzed by Becton Dickinson FACScan flow cytometry, and the sub-G1 population was quantified by WinMDI 2.9.

Microarray

LNCaP cells treated with mock and the 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene inhibitor for 3 days were harvested and the total RNA was extracted using Trizol reagent (Life Technologies, N.Y.). Microarray analysis was performed by the University of California Davis Cancer Center Gene Expression Resource, using Affymetrix Human Genome U133A (HG-U133A) GeneChip arrays (Affymetrix, Calif.), which permit expression analysis of the entire Genbank RefSeq database. Array scanning and generation of raw signal data files were done with GeneChip Operating Software (Affymetrix). Subsequent data analysis was done by GeneSpring (Agilent Technologies, Calif.) and DAVID Bioinformatic Resources 6.7 (NIH).

RESULTS :

KDM4B.PD2.H3K9me3 Crystal Structure

In an effort to understand the detailed structure-function relationship of KDM4B at an atomic resolution, the recombinant JmjC domain of KDM4B was subjected to crystallization in the presence of a peptide, Ni(II), and AKG or PD2. After extensive trials, a well-diffracting crystal was found to consist of a large piece of residual density in the binding pocket, which could be modeled as the H3K9me3 peptide, an inhibitor PD2 and Ni (FIG. 1A). The final crystal structure shows a 1.87 Å-resolution monomer (R=21.8%, R_(free)=26.2%) that consisted of the KDM4B catalytic domain (residues 9-337), PD2, and an H3K9me3 peptide (residues 7-14) within the active site (Table 1). A Ni(II) ion was observed to be located on a site corresponding to the Fe(II) position on the bottom of the catalytic pocket. The JmjC domain of KDM4B folded into a β-barrel structure, characteristic of members in the KDM4 family (FIG. 1B). Superposition of KDM4A.OGA.H3K9me3 (PDB code: 2OQ6), KDM4B.PD2.H3K9me3, KDM4C.OGA (PDB code: 2XML), and KDM4D.AKG.H3K9me3 (PDB code: 4HON) showed limited conformational change in overall Cα atoms of the JmjC domain. RMSD between KDM4A and KDM4B was 0.54 Å (residues 9-337 of KMD4B).

In the active site, PD2 was situated at a position nearly overlapped with AKG in which one of its carboxyl moieties contacted with H189, E191 and K242, while the other made H bonds with Y133 and K207, similar to those that contacted with AKG (Y132, N198, and K206 in KDM4A). Ni(II) that occupied the site of Fe(II) made contacts with three strictly conserved residues (H189, E191, and H277 in KDM4B). G171, Y176, T290, and N291 in KDM4B that surround the methylated lysine were also strictly conserved (FIG. 10).

The most prominent feature of the KDM4 family was its potent catalytic activity toward H3K9me3/me2. Analysis of superimposed H3K9me3 liganded structures including KDM4A, KDM4B, and KDM4D [KDM4A.Fe(II).OGA.H3K9me3 (PDB code: 2OQ6), KDM4B.Ni(II).PD2.H3K9me3 (this study; PDB code: 4LXL), KDM4D.Ni(II).2-OG.H3K9me3 (PDB code: 4HON)] revealed a conserved region to accommodate R8 (−1) and the methylated K9 of H3. Notably, three conserved residues (KDM4A: D135, E169, Y175; KDM4B: D136, E170, Y176; KDM4D: D139, E173, Y179) from β7 and β8 made H contacts with the guanidinium group of R8, the peptide O and N atoms of K9 and the peptide N atom of T11 from H3. In the interior of this cleft, a lysyl side chain (KDM4A: K241; KDM4B: K242; KDM4D: K245) forms a strong bond to the peptide O atom of S10 (FIG. 10), hence together properly orienting the H3K9me3/me2 for similarly efficient catalysis in KDMs. Interestingly, the KDM4A/KDM4B/KDM4C subfamily but not KDM4D exhibited additional specificity to demethylate H3K36me3/me2. Consistent with the structural analysis for KDM4D, the present invention observed two heterogeneous regions in KDM4B accounting for the substrate specificity: (1) RKDM vs. GEAR and (2) NIQ vs. HKK (FIG. 2).

The RKDM site (residues 310-313 in KDM4B) from a long U-shaped loop resided near the (−1, −2) site of the peptide-binding cleft. The aspartate side chain of RKDM (D311 in KDM4A) faced toward the peptide (−1, and −2 sites) and could make contacts with the peptide (FIG. 2). Additionally, the long and positively charged side chain of R from RKDM contributed to contact with the plus side of the peptide as demonstrated in two liganded structures: KDM4A.AKG.H3K9 (PDB code: 2Q8C) [KDM4A/R309 (NH1)-H3/G12 (O): 3.8 Å] and KDM4A.N-oxalylglycine.H3K36 (PDB code: 2P5B) [KDM4A/R309 (NH1)-H3/H39 (N): 3.8 Å]. A subtle difference was also noted at the other side of this U loop between KDM4A and KDM4B; there was a T308-D236 contact in KDM4B but not in KDM4A (the corresponding residues are S307 and E235). In contrast, the GEAR motif deviated away from the peptide-binding cleft, hence no contacts with the peptide.

The other region was NIQ site from the β4-β5 segment shared in KDM4A/KDM4B/KDM4C (residues 87-89 in KDM4B) while KDM4D had HKK at the corresponding region. Q89 was noted to contact with H3H39 and R40 (+3 and +4), whereas KDM4D consisted of HKK with positively charged side chains at the corresponding site (FIG. 2), which was likely to yield steric hindrance and electrostatic repulsion against H39 and R40 of H3K36me3. 171 that was nearby NIQ (KDMA, 171; KDM4B, 172) also played a crucial role (Krishnan and Trievel, 2013).

The present invention used the formaldehyde dehydrogenase (FDH)-coupled continuous fluorescent demethylase method to assess the enzymatic activity of the recombinant KDM4A and KDM4B expressed in Escherichia coli. Using an H3K9me3 peptide (residues 3-17) as the substrate, KDM4A and KDM4B exhibited comparable catalytic activity (Table 2), consistent with Hillringhaus et al. The present invention were able to measure the kinetic parameters with an H3K36me3 peptide (H331-41K36me3) and obtained an analogous k_(cat) value while a higher K_(m) value as compared with those with the H3K9me3 peptide, suggesting that KDM4A/4B had a lower binding affinity toward H3K36me3 than H3K9me3.

TABLE 2 Kinetic parameters for KDM4A and KDM4B using H3₃₋₁₇K9me3 or H3₃₁₋₄₁K36me3 as the substrate. KDM4 k_(cat) (s⁻¹) K_(m) (μM) k_(cat)/K_(m) (s⁻¹μM⁻¹) H3₃₋₁₇K9me3 KDM4A 0.017 ± 0.001 92.5 ± 5.9 1.8 × 10⁻⁴ KDM4B 0.014 ± 0.001 88.3 ± 8.6 1.6 × 10⁻⁴ H3₃₁₋₄₁K36me3 KDM4A 0.015 ± 0.001 169.9 ± 19.9 8.8 × 10⁻⁵ KDM4B 0.013 ± 0.001 138.5 ± 14.1 9.4 × 10⁻⁵

The present invention further utilized calf thymus histones as the substrate and probed for H3K9, H3K27 and H3K36me3/me2/me1 in the presence of the recombinant KDM4A or KDM4B using western blotting analysis. As shown in FIG. 11, the signal of H3K9me3/me2 was significantly reduced while that of H3K9me1 increased in the presence KDM4A (upper panel) or KDM4B (lower panel) via a time-dependent manner as compared with the controls, indicating an active KDM4A/KDM4B form to remove the methyl group from H3K9me3/me2. For H3K36 signal, a longer time was needed to remove the signal of H3K36me3/me2. No difference was found for H3K27me3 or H3K27me1. These results collectively suggested that KDM4A and KDM4B demethylated H3K9me3/me2 more efficiently than did H3K36me3/me2 and that there was no activity toward H3K27me3 /me2/me1, confirming the results in Table 2.

Virtual Screening to Identify 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene as a Novel Active-Site Inhibitor Toward KDM4A and KDM4B

The present invention utilized GEMDOCK to screen for putative hits against the NCI database. The known inhibitor PD2 was used as a positive control which showed significant inhibition [21% (KDM4A) and 24% (KDM4B) of residual activity]. Of the selected 10 top-ranked compounds, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene exhibited the highest inhibitory effect toward both KDM4A (28%) and KDM4B (35%) (Table 3). To confirm the FDH-demethylase coupled results, the present invention utilized histones as the substrate and probed for H3K9me3 and H3K36me3 in the absence or presence of the recombinant KDM4A or KDM4B using western blotting analysis. FIG. 3A shows that PD2 and 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene indeed blocked the demethylation activity. Further kinetic inhibition characterization of 1,5-bis[(E)-2-(3,4-dichlorophenyl)enthenyl]-2,4-dinitrobenzene demonstrated a competitive inhibitory mode against H3₃₋₁₇K9me3 for KDM4A [IC50=6.4 μM; Ki (H3K9me3)=5.5 μM; FIG. 3B). KDM4B also showed analogous inhibition kinetics [IC50=9.3 μM; Ki (H3K9me3)=3 μM). These results together provide strong evidence that 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene was a potent selective active-site inhibitor.

The De Novo Link protocol of Discovery Studio v3.0 (Accelrys Inc., USA) was used to suggest modifications and additions to the specific CL functional group of the docked NSC636819 in order to enhance binding to the KDM4B. This approach suggested modifications to a ligand scaffold to increase binding by placing fragments from the specified Ludi library into the specified binding site in accordance of the Ludi-generated interaction map.

The parameters used were as follows. Four CL atoms of the NSC636819 were defined as linked atoms with KDM4B as an input receptor and set an input sphere which includes whole Jmjc. Ludi-based fragment libraries were used here as input fragment libraries.

Then the simulated results showed that 54, 94, 11 and 3 for R1, R2, R3 and R4 groups respectively. The T01 (C₁₁H₁₀) fragment was illustrated as an example to show the enhanced binding capability due to additional cation-Pi interactions (See FIG. 14).

The KDM4B.1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene complex model

An in silico approach was used to generate KDM4B.1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene complex model by means of energy minimization and molecular dynamics simulations (FIG. 4). Given the model, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene was situated at a position that occupied across three sites: the AKG site (e.g., H189, E191, and H277), the key lysine binding region (e.g., D136, E170, Y176, K242), and a portion of the H3K36-specific NIQ region (e.g., 172, Q85, N87) (FIG. 4). Of note, the middle benzene ring with two nitro groups reached the terminal methylated N(ε) position as well as Fe(II) position. The other two 1,2-dichlorobenzene rings were situated at the (0) and (+2) sites of the peptide-binding cleft, thereby specifically inhibiting KDM4A and KDM4B activity. Thus, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene represented a novel KDM4A and KDM4B-specific inhibitor.

The present invention next compared the interactions of all tested compounds based on their docking poses. Three groups were classified: group 1 included all three sites (AKG, the methylated lysine, and NIQ); group 2 included AKG and the NIQ sites; and group 3 consisted of AKG and the methylated lysine sites (FIG. 5). Overall, compounds in group 2 and group 3 had low to modest inhibitory effects compared with 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene from group 1, suggesting the positive contribution of contacts from all three sites (FIG. 5). Above all, the meta-positioned NO₂ moieties on the benzene ring that occupied at the AKG and the methylated lysine sites (S179, H277, H189, and N199) were found for 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene that possessed inhibitory activity. In support of this notion, NSC26820 that had ortho-positioned COOH moieties completely lost the inhibitory effects (Table 3). In addition, NSC107408 showed no inhibition because it lacked the 1,3-dinitrobenzene moiety and was unable to form interactions with residues S 197, H277, H189, and N199 of the AKG site (FIGS. 5A and 5B).

TABLE 3 Inhibition effects of selected hits on KDM4A and KDM4B. Demethylase^(a) FDH^(b) Compound Residual IC₅₀ Residual Compound Ranking ID KDM Activity (%) (μM) Activity (%) structure 4B NSC636819 4A 4B 28 35 15.5  9.3 >80

100 NSC107408 4A 4B 99 64 — — >80

3 NSC85021 4A 4B 82 65 — — >80

223 NSC28620 4A 4B 111 110 — — >80

271 NSC15975 4A 4B 73 90 — — >80

83 NSC640999 4A 4B 77 49 — — >80

2 NSC659196 4A 4B 92 56 — — >80

33 NSC124029 4A 4B 93 62 — — 97

159 NSC1018 4A 4B 85 62 — — >80

93 NSC6129 4A 4B 100  52 — — >80

62

Genetic and pharmacological inhibition of KDM4A and KDM4B induced apoptosis

Several studies have reported that KDM4 family members are over-expressed in various cancers. To further support clinical relevance of KDM4A and KDM4B in prostate cancer, the present invention took advantage of the comprehensive database collection on Oncomine™ (Compendia Bioscience, Ann Arbor, Mich., USA) database to examine their expression profile between normal prostate gland and tumor tissues. Among 14 datasets available, a statistically significant (p<0.05) elevation of KDM4A was seen for 6, and KDM4B for 8 in PCa compared to norma;/benign samples (FIGS. 6A and 6B). The rest of datasets also exhibited higher median values of KDM4A/KDM4B expression in PCa sites, despite no statistical significance (FIGS. 13A and 13B). Strikingly, the level of KDM4A and B's expression was positively correlated with prostate cancer progression (normal, primary PCa, and metastatic PCa).

The present invention also examined the expression of KDM4A and KDM4B in several laboratory-cultured prostate cancer cell models: normal prostate epithelial cells (PrEC, RWPE-1 and PNT2) and a number of prostate cancer cell lines (LNCaP, C4-2, C4-2B, CWR22rv1, CWR-R1, VCap, DU145 and PC3). Essentially all prostate cancer cells exhibited higher expression of KDM4A as compared with normal prostate epithelial cell lines, in which statistical significance was found for LNCaP, C4-2, C4-2B, CWR22rv1, and VCap cells (FIG. 6C). Similarly, with the exception of DU145 and PC3, KDM4B was over-expressed in all other malignant cell lines.

To assess whether KDM4A or KDM4B was crucial for prostate cancer cell growth, LNCaP cells were treated with shKDM4A or shKDM4B to knockdown the expression of KDM4A or KDM4B (FIG. 7A). A significantly reduced level of cell growth was found in KDM4A and KDM4B knockdown cells (FIG. 7A), indicating that these two molecules were critical to the viability of the cancer cells and thus were potentially useful targets for intervention.

Due to the shKDM4A and 4B knockdown data whether 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene, which inhibited both KDM4A and 4B would similarly reduce the viability of LNCaP was tested. As shown in FIG. 7B, this compound effectively killed LNCaP cells with a cytotoxicity IC50 of 21.2 μM. Cell flow cytometric analysis showed that there were nearly four-fold apoptotic LNCaP cells produced upon 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene treatment as compared to mock cells (11.9% vs. 39.5%) (FIG. 7C). To validate the effect of 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene, the present invention examined the cellular level of H3K9me3 of the treated cells. As shown in FIG. 7B, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene-treated cells had a notable increase in the level of H3K9me3 in a dose-dependent manner; 5 μM of 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene treatment essentially completely blocked the demethylating activity toward H3K9me3. These results collectively suggested that inhibition of KDM4A/KDM4B by shRNA or by 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene specifically inhibited the demethylating activity of H3K9me3 and strongly blocked cell growth. As a comparison, the present invention utilized the dimethyl ester form of PD2, the most potent inhibitor against KDM4E (in vitro assay), allowing to penetrate into the cells. Consistent with their results, a high CC50 value was found in LNCaP cells (588.7 μM).

Inhibition of KDM4 by 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene negatively regulateed AR responsive genes

To understand the mechanisms associated with growth inhibition and apoptosis induction by 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene, the present invention characterized the differential gene expression profiles in LNCaP cells treated with or without 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene using microarray analysis (≧two-fold alterations). As shown in FIG. 8A, functional annotations indicated a number of differentially expressed genes related to cell division and DNA processes (Bar graph and the numbers labeled indicate gene count of each pathway). GO terms associated with the altered genes that showed statistically strong enrichment with low P-values were listed. % of hits indicated the percentage of genes that were altered in each GO (Table 4).

TABLE 4 The data of the GO term Total genes in Index P-Value each GO term % of hits a 2.32E−48 776 16 b 8.58E−45 220 31 c 2.65E−11 566 10 d 2.71E−09 485 10 e 3.91E−09 284 12 f 8.23E−08 98 18 g 1.13E−06 117 15 h 1.07E−05 436 8 i 4.63E−05 217 10 j 1.10E−04 857 6 k 9.51E−04 466 6 l 5.00E−03 105 10 m 5.90E−03 815 6 n 9.09E−03 564 6 o 1.23E−02 622 6

Most intriguingly, a significant portion (27%=178/656) of the altered genes were found to be androgen responsive genes (FIG. 8C). The mRNA expression of AR signature genes was validated using qRT-PCR (FIG. 9). This is consistent with the previous results that both KDM4A and KDM4B (as well as KDM4C) are coactivators of AR. In addition to the alteration of androgen responsive genes, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene induced up-regulation of tumour suppressors RB1, CDH1; as well as down-regulation of oncogenes IGF1R, FGFR3, CCNE2, AURKA and AURKB (FIG. 8B) which might contribute to the loss of proliferating and survival advantages for the tumour cell. Thus, 1,5-bis[(E)-2-(3,4-dichlorophenyl)ethenyl]-2,4-dinitrobenzene specifically inhibited the expression of genes involved in DNA-dependent processes, cell proliferation and AR-dependent signaling in prostate cancer cells. Given the importance of AR in prostate carcinogenesis, compounds inhibited KDM4A and 4B could be beneficially used to overcome castration-resistance.

The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, which are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims. 

What is claimed is:
 1. A method for identifying a compound which inhibits an activity of a histone lysine demethylase, comprising: (a) using a computer program to generate a three-dimensional structure of a pocket of a histone lysine demethylase, wherein the pocket comprises three sites: an alpha-ketoglutarate (AKG), a methylated lysine, and a NIQ; (b) screening for a compound that interacts with the three sites of said pocket; and (c) testing the compound screened in (b) by in vitro or in vivo assay for its ability to inhibit the activity of the histone lysine demethylase, thereby identifying a compound that inhibits the activity of the histone lysine demethylase.
 2. The method of claim 1, wherein the pocket of the histone lysine demethylase is formed by residues including Gln85, Tyr133, Asp136, Tyr176, Tyr178, Phe186, His189, Glu191, Asn199, Lys207, His241, Lys242, His277 and Asn291, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO:
 11. 3. The method of claim 1, wherein the pocket of the histone lysine demethylase is formed by residues including Gln84, Tyr132, Asp135, Tyr175, Tyr177, Phe185, His188, Glu190, Asn198, Lys206, His240, Lys241, His276 and Asn290, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO:
 12. 4. The method of claim 1, wherein the pocket of the histone lysine demethylase is formed by residues including Gln86, Tyr134, Asp137, Tyr177, Tyr179, Phe187, His190, Glu192, Asn200, Lys208, His242, Lys243, His278 and Asn292, wherein the amino acid position refers to the full length histone lysine demethylase shown in SEQ ID NO:
 13. 5. The method of claim 1, wherein the activity is the demethylating activity. 